The focus of this proposal is to investigate the role of the PERK eIF2a kinase (EIF2AK3) in regulating proinsulin trafficking and quality control in the endoplasmic reticulum. PERK is among a small set of genes identified in mice and humans that are so critically important to beta cell functions that loss of function mutations result in permanent neonatal diabetes due to insulin insufficiency. Moreover, mutations in Perk are the most common cause of consanguineous permanent neonatal diabetes in man. Our investigation of global and tissue-specific Perk KO mice led to the discovery that PERK is required for normal beta cell proliferation, proinsulin trafficking, and insulin secretion. However, the severity and magnitude of the beta cell defects seen when PERK expression is entirely absent makes the Perk KO unsuitable as the primary tool to discover the molecular and physiological functions of PERK in beta cells. We have chosen an alternative strategy whereby we will modulate PERK expression from 0.2 to 2-fold of normal levels and then investigate the acute effects on proinsulin trafficking and insulin secretion. A key assumption of this strategy is that more modest changes in expression can alter physiological outcomes. In support of this assumption we have already shown that reducing Perk gene dosage in half results in reduced blood glucose levels and ameliorates the progression of diabetes of the Akita insulin mutant mouse, whereas increasing Perk dosage above normal results in increased blood glucose levels and accelerates the progression of diabetes in Akita mice. We propose that PERK is required to regulate proinsulin quality control and trafficking in the endoplasmic reticulum as a function of the physiological demand for insulin. Hence our overall strategy is to test the this hypothesis by determining how PERK regulates quality control and trafficking of wildtype and Akita proinsulin, determine how PERK regulates ER chaperone and folding proteins underlying these functions, and determine how the regulation of PERK activity responds to normal changes in metabolism to mediate changes in proinsulin trafficking and insulin secretion. PUBLIC HEALTH RELEVANCE: Over the past several decades the number of cases of diabetes mellitus has grown significantly and is now one of our nation's leading causes of death and disability. Insufficient insulin synthesis and secretion contributes to all forms of diabetes mellitus in humans, so understanding how these processes work is critical if we are to continue to help patients manage this disease and eventually find a cure. The aims of this proposal are to characterize how a key gene in regulating functions of the insulin-secreting beta cell controls the synthesis and secretion of insulin.